Calpain activation in apoptosis of ventral spinal cord 4.1 (VSC4.1) motoneurons exposed to glutamate: Calpain inhibition provides functional neuroprotection

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca²⁺ influx may mediate activation of calpain, a Ca²⁺-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 μM glutamate for 24 hr caused significant increases in intracellular free [Ca²⁺], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 μM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca²⁺ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca²⁺ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.